Role of Parathyroid Hormone in the Downregulation of Liver Cytochrome P450 in Chronic Renal Failure
Josée Michaud*,,
Judith Naud*,,
Jérôme Chouinard*,
François Désy*,
Francois A. Leblond*,
Karine Desbiens*,
Alain Bonnardeaux* and
Vincent Pichette*,
* Service de néphrologie et Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, and Département de Pharmacologie, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada
Address correspondence to: Dr. Vincent Pichette, Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boulevard de l’Assomption, Montréal, Québec, Canada, H1T 2M4. Phone: 514-252-3489; Fax: 514-255-3026; E-mail: vpichette.hmr{at}ssss.gouv.qc.ca
Received for publication January 13, 2006.
Accepted for publication August 21, 2006.
Chronic renal failure (CRF) is associated with a decrease indrug metabolism secondary to a decrease in liver cytochromeP450 (P450). The predominant theory to explain this decreaseis the presence of factors in the blood of uremic patients.This study tested the hypothesis that parathyroid hormone (PTH)could be this factor. The objectives of this study were to determine(1) the role of PTH in the downregulation of hepatocyte P450induced by rat uremic serum, (2) the role of PTH in the downregulationof liver P450 in rats with CRF, and (3) the effects of PTH onP450 in hepatocytes. For this purpose, (1) hepatocytes wereincubated with serum from rat with CRF that was depleted withanti-PTH antibodies or with serum from parathyroidectomized(CRF-PTX) rat with CRF, (2) the effect of PTX on liver P450was evaluated in rats with CRF, and (3) the effects of PTH onP450 in hepatocytes were determined. The depletion of PTH fromCRF serum completely reversed the downregulating effect of CRFserum on P450 in hepatocytes. Addition of PTH (10–9 M)to depleted CRF serum induced a decrease in P450 similar tonondepleted CRF serum. The serum of CRF-PTX rats had no effecton P450 in hepatocytes compared with CRF serum. Adding PTH toCRF-PTX serum induced a similar decrease in P450 as obtainedwith CRF serum. Finally, PTX prevented the decrease of liverP450 in rats with CRF. In summary, PTH is the major mediatorimplicated in the downregulation of liver P450 in rats withCRF.
Chronic renal failure (CRF) interferes with the eliminationof many drugs because of the reduction in GFR and tubular secretion(1). However, renal failure also diminishes the metabolic clearanceof selected drugs secondary to decrease of hepatic and intestinalmetabolism of these drugs (2–6). The major determinantfor these metabolic changes is a reduction in enzymatic activity.
Cytochrome P450 (P450) is the major catalyst of drug biotransformation.Several animal studies have shown that liver and intestinalP450 are reduced in CRF (7–10). These studies demonstratedthat CRF is associated with a decrease in the activity as wellas in the expression of liver and intestinal P450 isoforms secondaryto reduced mRNA levels (9,10). The main hypothesis to explainP450 activity and expression downregulation is the presencein the blood of uremic animals of endogenous inhibitors thatmodulate the P450. Indeed, we have shown that in normal hepatocytesthat were incubated for 24 h with serum from rats with CRF,total P450 level and protein expression of several P450 isoformsdecreased by 45% compared with serum from control animals (11).This decrease in protein expression of P450 isoforms was secondaryto reduced gene expression (11). Similar results have been shownwith serum of patients with severe CRF (12). The next step wasto find which factor in the uremic blood downregulates P450in CRF.
CRF is associated with multiple metabolic disturbances. As aconsequence, numerous molecules are increased in CRF. However,taking into account the changes that are induced by CRF (metabolic,hormonal, and retention of toxins) and the factors that areknown to affect the P450, two main mediators are most likelyto be associated with downregulation of P450 in CRF: parathyroidhormone (PTH) and proinflammatory cytokines (6). Although thepotency of cytokines to downregulate P450 have been establishedin inflammatory disease (13), we hypothesized that PTH couldbe implicated in the downregulation of P450 in CRF for the followingreasons: (1) secondary hyperparathyroidism is frequent in CRF(14); (2) PTH is known to downregulate the mRNA of many proteins,particularly in the liver but also in other tissue such as theheart (15–17); and (3) we have found a strong correlationbetween the levels of PTH and P450 reduction induced by serumof patients (12).
The objectives of this study were to determine (1) the roleof PTH in the in vitro downregulation of liver P450 that isinduced by rat uremic serum, (2) the role of PTH in the in vivodownregulation of liver P450 in CRF rats, and (3) the in vitroeffects of PTH on P450 in cultured hepatocytes. For this purpose,(1) we incubated normal rat hepatocytes with CRF rat serum thatwas preadsorbed on immobilized anti-PTH antibodies or with serumfrom CRF parathyroidectomized (CRF-PTX) rat, (2) we evaluatedthe effect of PTX on liver P450 activity and expression in ratswith CRF, and (3) we determined the effects of PTH on P450 incultured hepatocytes.
Experimental Model
Male Sprague-Dawley rats (Charles River, Saint-Charles, QC,Canada) that weighed 200 to 300 g were housed in the ResearchCentre animal care facility and maintained on Purina rat pellets(Ralston-Purina, St. Louis, MO) and water ad libitum. An acclimatizationperiod of 3 d was allowed before any experimental work was undertaken.All of the experiments were conducted according to the CanadianCouncil on Animal Care guidelines for care and use of laboratoryanimals.
As shown in Table 1, rats were divided in four groups. Hepatocyteswere isolated from normal rats (n = 35), and the sera that wereused for incubation experiments were obtained from CRF, CRF-PTX,control, or control-PTX rats at the time of sacrifice.
CRF was induced by two-stage five-sixths nephrectomy as previouslypublished (9,18). After surgery, rats with CRF were fed Purinarat chow and water ad libitum. Rats from the control group alsounderwent two sham laparotomies (days 1 and 8). Control ratswere pair-fed the same amount of rat chow that was ingestedby the rats with CRF on the previous day. At day 41 after thenephrectomy, the rats were housed in metabolic cages and urinewas collected for 24 h to determine the clearance of creatinine.Rats were killed by decapitation at 42 d. Blood was collectedand stored rapidly on ice. After coagulation, serum was recoveredby centrifugation (600 x g for 10 min at 4°C), and sampleswere kept for the measurement of serum creatinine and urea.The remaining sera were stored at –80°C.
Total parathyroidectomy (PTX) was performed as described previously(16). Briefly, surgical PTX was carried out under a surgicalmicroscope, without removal of the thyroid tissue. The successof the PTX was ascertained by a significant decrease of calciumafter PTX. To avoid hypocalcemia, PTX rats then were supplementedin calcium by addition of calcium gluconate to drinking water(control 5%; CRF 2.5%). Rats then were allowed to recover for1 wk before the five-sixths nephrectomy. Control rats receivedsham surgery in the neck region.
Immunoadsorption of PTH in Uremic Serum
To deplete PTH in the sera of rats with CRF, we used polystyrenebeads (8.4 mm) coated with goat antibodies that were specificto the N-terminal (1-34) region of rat PTH (Alpco Diagnostics,Windham, NH). Each of these beads can bind at least 400 pg ofPTH. Rat sera were incubated at 4°C for 16 h with beads(7 beads/ml serum). After depletion, treated sera were addedto culture medium (10%) and filtered on 0.22-µm filter.Samples of depleted sera were reserved for quantification ofPTH after depletion. Nondepleted control and CRF sera were incubatedconcomitantly at 4°C and treated in the same way.
Hepatocyte Isolation and Culture
Hepatocytes were isolated from normal rats according to thetwo-step liver perfusion method of Seglen as previously published(11). Collagenase type 4 (Worthington, Lakewood, NJ) was used.
After preincubation, the medium was changed for 2 ml of WilliamE medium that contained 10% of serum from rats with CRF, CRF-PTX,or control rats. The serum of one rat was used for one experiment.Thereafter, the hepatocytes were incubated for another 24 h.Hepatocytes then were harvested by scraping in PBS. For mRNAanalysis, cells were harvested in RLT buffer (Qiagen, Mississauga,Ontario, Canada). Samples were stored at –80°C untilanalysis.
For assessment of whether liver P450 could be downregulatedby PTH, a dosage-response curve was obtained by measurementof the ability of a wide-range PTH (rat synthetic 1-34 PTH;Sigma, St. Louis, MO) concentrations (10–12 to 10–7M) to depress the P450 of normal hepatocytes. Incubation timewas 24 h. PTH was dissolved in 0.15 N acetic acid. Hepatocytesthen were harvested as described in the previous paragraph andstored at –80°C until analysis.
Effect of Inhibition of the NF-B Pathway on PTH-Induced Downregulation of Liver CYP3A2
For assessment of whether the blockade of the NF-B pathway couldprevent the effect of PTH on liver P450, two inhibitors of NF-Bwere used: pyrrolidine dithiocarbamate (PDTC) (19) and Andrographolide(20) (Calbiochem, San Diego, CA). Normal hepatocytes were incubatedfor 24 h in the presence of either PDTC (50 µM) or Andrographolide(30 µM) or in absence of these inhibitors with and withoutPTH (10–9 M).
Microsome Preparation from Hepatocytes and Liver
Hepatocyte microsomes were isolated by differential centrifugation(21). The pellet that contained the microsomes was resuspendedin 0.9% NaCl and stored at –80°C until analysis. Livermicrosomes were isolated by differential centrifugation accordingto Cinti et al. (22). The pellet that contained the microsomeswas stored at –80°C in 0.1 M Tris (pH 7.4), 20% glycerol,and 10 mM EDTA until analysis.
Western Blot Analysis
Although several cytochrome P450 isoforms are implicated inthe metabolism of drugs, we assessed only CYP1A1, CYP2C11, CYP2E1,and CYP3A2, which are the more abundant isoforms in the ratliver and are most affected by CRF as previously reported (9).These isoforms were assessed by Western blotting as describedelsewhere (9). P450 were detected using polyclonal goat anti-rat1A1, 2C11, 2E1, and 3A2 (Gentest Corp., Woburn, MA), respectively.-Actin was detected using a mouse anti-chicken -actin (Neo-Markers,Fremont, CA). Immune complexes were revealed by secondary antibodies(swine anti-goat IgG coupled to peroxidase [Biosource International,Camarillo, CA] or goat anti-mouse IgG coupled to peroxidase[Sigma]) and the Luminol derivative of Lumi-Light Western blottingsubstrate (Roche Diagnostics, Laval, Quebec, Canada). Immunereaction intensity was determined by computer-assisted densitometryon Fuji (Stamford, CT) LAS-3000 LCD camera coupled to the analysisprogram MultiGauge (Fuji).
RNA Isolation and Real-Time Quantitative PCR Analysis
RNA extractions were done on either the liver or the hepatocyteswith the RNeasy Midi and Mini Kit (Qiagen), respectively. Onemicrogram of total RNA was used to prepare cDNA by reverse transcriptionusing Omniscript RT kit (Qiagen) and random primer (Invitrogen,Burlington, Ontario, Canada). Quantitative PCR analysis wasperformed using Platinum SYBR green qPCR (Invitrogen) on theiCycler real-time detection system (Bio-Rad Laboratories, Mississauga,Ontario, Canada). Specific primer sets were designed for eachof the two tested mRNA (3A2 and glyceraldehyde-3-phosphate dehydrogenase)on the basis of published cDNA sequences with the aid of theJellyfish computer program (LabVelocity Inc., Los Angeles, CA)and are reported in Table 2. All primers were obtained fromSigma, and their specificity was confirmed by sequencing ofthe resulting PCR product on ABI Prism 3100 analyzer (AppliedBiosystems, Foster City, CA). Used PCR conditions were optimizedto 95°C for 15 s, 59°C for 30 s, and 72°C for 60s. The respective PCR products were cloned in the pCr 2.1 vectorusing TA cloning Kit (Invitrogen). The resulting plasmids werepurified with Hispeed Plasmid Midi Kit (Qiagen), quantifiedat 260 nm and diluted to make a standard curve.
Evaluation of CYP3A Activity
For evaluation of the metabolic activity of CYP3A in microsomeof either treated hepatocytes or the livers microsomes of thedifferent groups of rats, a selective fluorescence probe, 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl]furan-2(5H)-one(DFB), which is specifically metabolized by rat CYP3A2, wasused as previously reported (23). In the case of microsomes,25 µg of protein was used. The fluorescence of the metabolite3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one(DFH) was read on the cytofluorometer (Cytofluor 4000/TR; PerspectiveBiosystems, Framingham, MA) using appropriate wavelength (excitationfilter 360/40 nm; emission filter 460/40 nm). Standard curvewas prepared with known dilution of DFH. For hepatocytes, substratewas added directly to the culture after removal of the culturemedium and replacement by Krebs buffer that contained 12.5 mMHEPES.
Blood and Urine Chemistries
Blood (urea, creatinine, calcium, and phosphate) and urine (creatinine)chemistries were determined with a Hitachi 717 autoanalyzer(Roche). PTH was measured by using the Rat intact PTH ELISAKit (Alpco Diagnostics), which measure the intact 1-84 PTH.The lowest detectable level is 15 pg/ml.
Statistical Analyses
The results are expressed as mean ± SEM. Differencesbetween groups were assessed by using an unpaired t test oran ANOVA test. Significant ANOVA was followed by a post hocScheffe analysis (Dunnett for Figure 4). The threshold of significancewas P < 0.05.
Figure 4. Dose-response effect of PTH on CYP3A2 protein () and mRNA expression () by hepatocytes incubated for 24 h with various concentrations of 1-34 rat PTH in 10% calf serum. CYP3A2 protein expression was measured by Western blot; specific mRNA were quantified by qPCR. Results of samples incubated with calf serum without added PTH were defined as 100%. Representative blots were shown in insert. *P<0.01 when protein expression was compared with hepatocytes incubated without PTH; P<0.01 when mRNA expression was compared with hepatocytes incubated without PTH.
Biochemical Parameters and Body Weight in Rats Table 1 presents the biochemical parameters and body weightof the four groups of animals studied. Compared with controlrats, rats with CRF had higher levels of plasma creatinine andlower values of creatinine clearance, which was reduced by 80%(P < 0.001). Body weights in control rats and rats with CRFwere similar. The serum PTH was increased 10-fold in rats withCRF, reflecting the magnitude of secondary hyperparathyroidism.PTH levels were undetectable in PTX rats. Although, there wasa small decrease of plasma calcium in PTX rats, it was not statisticallydifferent from that in control rats (Table 1).
Role of PTH in the Downregulation of Liver P450 Induced by Uremic Serum
Two sets of experiments were conducted. We first neutralizedPTH in uremic serum. As shown in Figure 1, we confirmed ourprevious results as CRF serum induced a 34 and 43% decreasein CYP3A protein expression and mRNA levels, respectively. CRFserum that was depleted of PTH lost its inhibitory capacityon CYP3A expression and activity (Figure 1). PTH concentrationsin CRF serum were reduced by 85 to 99% by depletion with immobilizedanti-PTH. Addition of PTH (at a concentration similar to CRFserum, which is 10–9 M) into depleted CRF serum induceda similar decrease in CYP3A levels as nondepleted CRF serum,confirming that the depletion procedure was specific for PTH(Figure 1). Similar results were obtained for CYP3A activitythat was evaluated by the DFB assay. We also evaluated anothermajor isoform in the rat (CYP2C11), and as shown for CYP3A,the depletion in PTH of CRF serum reverses the downregulationof CYP2C11 that is induced by uremic serum (data not shown).
Figure 1. Expression of CYP3A2 protein (), mRNA (), and drug metabolizing activity as measured by using 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl]furan-2(5H)-one (DFB) as specific substrate () in normal rat hepatocytes incubated for 24 h with control or CRF rat sera. Sera were used either nontreated or after preadsorption during 16 h at 4°C with latex beads coated with antibodies specific for rat parathyroid hormone (PTH) to remove PTH. A portion of the depleted sera was supplemented with 10–9 M 1-34 rat PTH before incubation with hepatocytes. CYP3A2 protein expression was measured by Western blot; specific mRNA were quantified by quantiative PCR (qPCR). Results of samples incubated with control rat serum were defined as 100%. Representative blots were shown in insert. *P <0.05 when compared with control (CTL) serum; P <0.05 when compared with depleted CRF sera.
To confirm further the role of PTH in uremic serum, we evaluatedthe effect of serum from PTX rats. As shown in Figure 2, thesera of CRF-PTX rats have no downregulating effects on CYP3Aprotein and mRNA expression. Again, addition of PTH (10–9M) to CRF-PTX sera induced a similar decrease in CYP3A levelsas obtained with CRF sera (Figure 2). Similar results were obtainedwhen we evaluated the activity of CYP3A by the DFB assay andalso for the expression of CYP2C11 (data not shown).
Figure 2. Expression of CYP3A2 protein (), mRNA (), and drug metabolizing activity as measured by using DFB as specific substrate () in normal rat hepatocytes incubated for 24 h with control, CRF, or PTX-CRF rat sera. A portion of the PTX-CRF sera was supplemented with 10–9 M 1-34 rat PTH before incubation with hepatocytes. CYP3A2 protein expression was measured by Western blot; specific mRNA were quantified by qPCR. Results of samples incubated with control rat serum were defined as 100%. Representative blots were shown in insert. *P<0.05 when compared with CTL serum. P<0.05 when compared with PTX-CRF sera.
Role of PTH in the In Vivo Downregulation of P450 Induced by CRF
These experiments were to determine whether PTH is implicatedin the in vivo decrease of P450 that is found consistently inCRF. As shown in Figure 3, we confirmed that in CRF, there wasa 72% decrease in liver CYP3A protein expression. However, preventingthe development of secondary hyperparathyroidism by PTX partlyreverses the negative effect of CRF on CYP3A. The mRNA codingfor CYP3A2 was significantly reduced in rats with CRF. We alsoobserved a significant increase in mRNA levels in CRF-PTX rats.The magnitude of this increase was similar (approximately 300%)for both protein expression and mRNA. Similar results were obtainedwhen we evaluated the metabolic activity of CYP3A by the DFBassay.
Figure 3. Expression of CYP3A2 protein (), mRNA (), and drug metabolizing activity as measured by using DFB as specific substrate () in sham operated (CTL) or CRF rats with or without prior parathyroidectomy (PTX). CTL rats were defined as 100% for CRF rats. Similarly, CTL-PTX were defined as 100% for CRF-PTX rat. CYP3A2 protein expression was measured by Western blot. Representative blots were shown in insert. *P<0.001 when compared with CTL rats; P<0.05 when compared with CRF rats; P <0.05 when compared with CTL-PTX rats.
In Vitro Effect of PTH on P450 in Cultured Hepatocytes
To confirm that PTH not only was implicated in the downregulationof liver P450 in uremia but also could regulate in vitro liverP450, we incubated during 24 h normal hepatocytes with synthetic1-34 rat PTH.
Dosage-Response Curve of PTH on Liver CYP3A in Cultured Hepatocytes. Figure 4 depicts the effects of various concentrations of PTH(10–12 to 10–7 M) on the protein expression as wellas on the mRNA levels of CYP3A2 of hepatocytes. As the concentrationof PTH increased, there was a dosage-dependant decrease in CYP3A2levels at both the protein and the mRNA level that tended toplateau at 10–8 M. It is interesting that the in vivoconcentrations that were obtained in our rats with CRF werebetween 10–10 and 10–9 M (see Table 1). There wasno effect of PTH on other control hepatocyte proteins (glyceraldehyde-3-phosphatedehydrogenase, aspartate aminotransferase, or -actin; data notshown).
Effects of PTH on CYP3A Activity in Cultured Hepatocytes.
The effects of PTH (at a concentration of 10–9 M) on theCYP3A activity are shown on Figure 5. When hepatocytes wereincubated with PTH, the activity of CYP3A was decreased by approximately40%, which is a decrease similar to that obtained with uremicserum or in rats with CRF (Figures 1 and 2).
Figure 5. Effect of PTH 10–9 M on the CYP3A2 metabolic activity of 24-h cultured hepatocytes. Metabolic activity was measured with DFB as specific substrate. Washed cells were incubated for 60 min with DFB at 37°C. Metabolite (DFH) production was measured on supernatant by fluorometry. *P <0.001 when compared with hepatocytes incubated without PTH, which were defined as 100%.
Effects of PTH on Other P450 Isoforms.
Because CRF as well as uremic serum was associated with a decreasein several P450 isoforms, we were interested to determine whetherPTH could modulate other P450 isoforms in cultured hepatocytes.Figure 6 shows that besides CYP3A, PTH (10–9 M) coulddecrease the protein expression of CYP1A1 and CYP2C11 and hadno effect on CYP2E1.
Figure 6. Effect of PTH on the protein expression of other P450 isoforms in hepatocytes incubated for 24 h with 10–9 M rat PTH (). Specific isoforms were determined by Western blot using monospecific antibodies. Expression of each isoform in hepatocytes incubated without PTH () was defined as 100%. Representative blots were shown in insert. *P<0.001.
Effects of NF-B Inhibitors on PTH-Induced Decrease of P450.
PDTC and Andrographolide are known to be specific inhibitorsof the NF-B pathway (19,20). Figure 7 shows that the decreasein CYP3A protein expression, mRNA, and activity by PTH (10–9M) was prevented by the addition of PDTC and Andrographolideto hepatocytes’ culture media.
Figure 7. Expression of CYP3A2 protein measured by Western blot (), and drug metabolizing activity measured by DFB assay () in normal rat hepatocytes incubated for 24 h with or without PTH in presence or absence of andrographolide or pyrrolidine dithiocarbamate (PDTC). CTL hepatocytes were defined as 100%. Representative blots were shown in insert. *P<0.001 when compared with CTL hepatocytes.
This study demonstrates that PTH is the major mediator implicatedin the downregulation of liver P450 in rats with CRF. Indeed,we showed that the depletion of CRF serum with anti-PTH antibodiescompletely reverses the inhibitory effect on P450 of CRF serumin cultured hepatocytes. Addition of PTH (10–9 M) to depletedCRF serum induced a similar decrease in P450 as nondepletedCRF serum. The serum of CRF-PTX rats had no effect on P450 comparedwith CRF serum. Again, addition of PTH to CRF-PTX serum induceda similar decrease in P450 as that obtained with CRF serum.Finally, PTX partly prevented the decrease of liver P450 inrats with CRF.
Renal failure generally has been thought to decrease only therenal clearance of drugs (24). However, several studies havedemonstrated that CRF also decreases hepatic drug metabolismsecondary to a decrease in liver P450 (9). The main reason fordecreased P450 activity and expression in the liver seems tobe the presence of uremic factors that accumulate in CRF (11).Indeed, we have shown that in normal hepatocytes that were incubatedfor 24 h with serum from rats or patients with CRF, total P450level and protein expressions of several P450 isoforms decreasedby 45% compared with serum from control animals (11,12). Thisdecrease in protein expression of P450 isoforms was secondaryto reduced gene expression (11,12).
In this study, we hypothesized that PTH could be a major factorin the downregulation of liver P450 that is induced by CRF,and our results confirm this hypothesis. Indeed, incubationof normal rat hepatocytes with CRF rat serum that was preadsorbedon immobilized anti-PTH completely reversed the inhibitory effectof uremic serum on cytochrome P450 (Figure 1). Similar resultswere obtained when we incubated serum from CRF-PTX rats (Figure 2).Furthermore, our results showed that PTH could downregulateliver P450 isoforms. Incubation of hepatocytes from normal ratswith rat PTH (1-34) resulted in a dosage-dependent reductionin the protein expression as well as in mRNA of CYP3A2 and 2C11(Figures 5 and 6). At concentrations that are found in CRF (10–10to 10–9 M), there was a reduction of >40% in the P450,similar to what we found with CRF serum.
CRF is associated with an increase in PTH (secondary or tertiaryhyperparathyroidism), which causes several uremic complications(25–29). More specific, PTH downregulates the mRNA ofmany proteins, such as hepatic lipase, as well as the receptorsfor vasopressin, angiotensin II in hepatocytes, and IGF-1 incardiomyocytes (15–17). Recently, CRF-induced resistanceto IGF-1 was attenuated by PTX, suggesting a role of PTH inthis resistance (17). The mechanisms underlying these effectsof PTH seem to be related to an increase of cAMP and/or an increasein intracellular calcium (26).
The effects of PTH on the isoforms of P450 that are implicatedin the metabolism of drugs, as found in this study, also couldbe secondary to activation of cAMP and increase in [Ca2+]i.PTH signaling pathway includes the generation of cAMP with activationof protein kinase A and subsequent phosphorylation of proteins(30). In hepatocytes, PTH increases cAMP production (31), whichcould cause a phosphorylation of P450 by a cAMP-dependent proteinkinase, leading to a decrease in the activity of selected isoenzymesof the P450 (32,33) or a downregulation of the expression ofgenes (CYP1A1, 2B1, 2B6, and 3A1) (34,35). However, some dataalso suggest that PTH may activate protein kinase C (30), andthat also could lead to P450 inhibition (36). However, the roleof [Ca2+]i in the regulation of P450 remains poorly defined(35,37,38).
Aside from these well-characterized PTH-signaling pathways,recent data suggest a third hypothesis through which PTH couldinhibit P450. This is via activation of the NF-B. NF-B is apleiotropic transcription factor that plays an important rolein the regulation of physiologic processes, including immuneresponses, inflammatory reactions, cell proliferation, apoptosis,and developmental processes (39). Several studies have shownthat NF-B plays an important role in mediating the suppressionof P450 expression by inflammatory agents, such as inflammatorycytokines and LPS (40,41). Moreover, it has been reported thatPTH and PTH-rP cause dosage- and time-related increases in NF-Bin human and rat osteoblastic cells (42,43). Our results supportthe hypothesis that PTH may act via NF-B to downregulate P450.Indeed, we have shown that the inhibition of the NF-B pathwayprevents the decrease in CYP3A2 expression and activity (Figure 7).Further studies are ongoing to identify the precise mechanismof PTH-induced P450 downregulation.
In this study, we also evaluated whether PTX could prevent thedownregulation of liver P450 that is induced in vivo by CRF.As shown in Figure 3, P450 decreased by 72% in liver of ratswith CRF but only by 28% in liver of CRF-PTX rats. This reduction,although less important, still was significant compared withcontrol rats. These results also suggest that PTH is a majorfactor not only in vitro but also in vivo but that in vivo,other factors could be implicated in the downregulation of liverP450. One of these factors could be cytokines. Indeed, severalstudies have demonstrated that CRF is associated with a chronicactivation of inflammatory response (44,45). Patients with CRFshow an increase in plasma levels of many cytokines, such asIL-1, monocyte chemotactic and activating factor, IL-6, granulocyteinhibitory protein, and TGF (46–50). However, cytokinesare able to downregulate hepatic P450 in vitro and in vivo (13).These observations support the hypothesis that, besides PTH,cytokines could downregulate the liver P450 in rats with CRF.
CRF is associated with a decrease in liver P450 secondary toreduced mRNA levels. The main reason for the decrease in P450is the presence of uremic factors that accumulate in CRF. Inthis study, we demonstrated that PTH is one of these factors.Preventing secondary hyperparathyroidism by PTX precludes thedownregulation of liver P450 in rats with CRF. Finally, we haveidentified a new hormone (PTH) that modulates the major drug-metabolizingsystem, the P450.
Acknowledgments
This work was supported by the Canadian Institutes of HealthResearch and the Fond de la Recherche en Santé du Québec.
Part of this work was presented at the 14th International Conferenceon Cytochromes P450; May 31 to June 5, 2005; Dallas, TX.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Matzke GE, Frey RF: Drug dosing in patients with impaired renal function. In:
Pharmacotherapy: A Pathophysiologic Basis, 2nd Ed., edited by Pipiro JT TR, Hayes PE, Yee GC, Matzke GR, Posey ML, New York, Elsevier Science Publishing, 1992
, pp 750
–763
Matzke GR, Frye RF: Drug administration in patients with renal insufficiency. Minimising renal and extrarenal toxicity.
Drug Saf 16
: 205
–231, 1997[Medline]
Touchette MA, Slaughter RL: The effect of renal failure on hepatic drug clearance.
DICP 25
: 1214
–1224, 1991[Abstract]
Gibson TP: Renal disease and drug metabolism: An overview.
Am J Kidney Dis 8
: 7
–17, 1986[Medline]
Pichette V, Leblond F: Metabolism of drugs in chronic renal failure.
Recent Res Dev Drug Metab Dispos 1
: 43
–56, 2002
Pichette V, Leblond FA: Drug metabolism in chronic renal failure.
Curr Drug Metab 4
: 91
–103, 2003[CrossRef][Medline]
Uchida N, Kurata N, Shimada K, Nishimura Y, Yasuda K, Hashimoto M, Uchida E, Yasuhara H: Changes of hepatic microsomal oxidative drug metabolizing enzymes in chronic renal failure (CRF) rats by partial nephrectomy.
Jpn J Pharmacol 68
: 431
–439, 1995[Medline]
Patterson SE, Cohn VH: Hepatic drug metabolism in rats with experimental chronic renal failure.
Biochem Pharmacol 33
: 711
–716, 1984[CrossRef][Medline]
Leblond F, Guevin C, Demers C, Pellerin I, Gascon-Barre M, Pichette V: Downregulation of hepatic cytochrome P450 in chronic renal failure.
J Am Soc Nephrol 12
: 326
–332, 2001[Abstract/Free Full Text]
Leblond FA, Petrucci M, Dube P, Bernier G, Bonnardeaux A, Pichette V: Downregulation of intestinal cytochrome p450 in chronic renal failure.
J Am Soc Nephrol 13
: 1579
–1585, 2002[Abstract/Free Full Text]
Guevin C, Michaud J, Naud J, Leblond FA, Pichette V: Down-regulation of hepatic cytochrome P450 in chronic renal failure: Role of uremic mediators.
Br J Pharmacol 137
: 1039
–1046, 2002[CrossRef][Medline]
Michaud J, Dube P, Naud J, Leblond FA, Desbiens K, Bonnardeaux A, Pichette V: Effects of serum from patients with chronic renal failure on rat hepatic cytochrome P450.
Br J Pharmacol 144
: 1067
–1077, 2005[CrossRef][Medline]
Abdel-Razzak Z, Loyer P, Fautrel A, Gautier JC, Corcos L, Turlin B, Beaune P, Guillouzo A: Cytokines down-regulate expression of major cytochrome P-450 enzymes in adult human hepatocytes in primary culture.
Mol Pharmacol 44
: 707
–715, 1993[Abstract]
Levi R, Silver J: Pathogenesis of parathyroid dysfunction in end-stage kidney disease.
Pediatr Nephrol 20
: 342
–345, 2005[CrossRef][Medline]
Massry SG, Klin M, Ni Z, Tian J, Kedes L, Smogorzewski M: Impaired agonist-induced calcium signaling in hepatocytes from chronic renal failure rats.
Kidney Int 48
: 1324
–1331, 1995[Medline]
Klin M, Smogorzewski M, Ni Z, Zhang G, Massry SG: Abnormalities in hepatic lipase in chronic renal failure: Role of excess parathyroid hormone.
J Clin Invest 97
: 2167
–2173, 1996[Medline]
Qing DP, Ding H, Vadgama J, Wu YY, Kopple JD: Elevated myocardial cytosolic calcium impairs insulin-like growth factor-1-stimulated protein synthesis in chronic renal failure.
J Am Soc Nephrol 10
: 84
–92, 1999[Abstract/Free Full Text]
Leblond FA, Giroux L, Villeneuve JP, Pichette V: Decreased in vivo metabolism of drugs in chronic renal failure.
Drug Metab Dispos 28
: 1317
–1320, 2000[Abstract/Free Full Text]
Xia YF, Ye BQ, Li YD, Wang JG, He XJ, Lin X, Yao X, Ma D, Slungaard A, Hebbel RP, Key NS, Geng JG: Andrographalide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50.
J Immunol 173
: 4207
–4217, 2004[Abstract/Free Full Text]
Munoz C, Pascual-Salcedo D, Castellanos MC, Alfranca A, Aragones J, Vara A, Redondo JM, de Landazuri MO: Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: Modulation of NF-kappa B and AP-1 transcription factors activity.
Blood 88
: 3482
–3490, 1996[Abstract/Free Full Text]
Tindberg N, Baldwin HA, Cross AJ, Ingelman-Sundberg M: Induction of cytochrome P450 2E1 expression in rat and gerbil astrocytes by inflammatory factors and ischemic injury.
Mol Pharmacol 50
: 1065
–1072, 1996[Abstract]
Cinti DL, Moldeus P, Schenkman JB: Kinetic parameters of drug-metabolizing enzymes in Ca2+-sedimented microsomes from rat liver.
Biochem Pharmacol 21
: 3249
–3256, 1972[CrossRef][Medline]
Nicoll-Griffith DA, Chauret N, Houle R, Day SH, D’Antoni M, Silva JM: Use of a benzyloxy-substituted lactone cyclooxygenase-2 inhibitor as a selective fluorescent probe for CYP3A activity in primary cultured rat and human hepatocytes.
Drug Metab Dispos 32
: 1509
–1515, 2004[Abstract/Free Full Text]
Cantu TG, Ellerbeck EF, Yun SW, Castine SD, Kornhauser DM: Drug prescribing for patients with changing renal function.
Am J Hosp Pharm 49
: 2944
–2948, 1992[Abstract]
Goltzman D, Hurdy G: Parathyroid hormone. In:
Principles and Practice of Endocrinology and Metabolism, edited by Becker KL, Philadelphia, Lippincott, 1990
, pp 402
–411
Massry SG, Smogorzewski M: Parathyroid hormone, chronic renal failure and the liver.
Kidney Int Suppl 62
: S5
–S7, 1997[Medline]
Smogorzewski M, Massry SG: Defects in B-cell function and metabolism in uremia: Role of parathyroid hormone.
Kidney Int Suppl 78
: S186
–S189, 2001[CrossRef][Medline]
Llach F, Velasquez Forero F: Secondary hyperparathyroidism in chronic renal failure: Pathogenic and clinical aspects.
Am J Kidney Dis 38
[Suppl 5]: S20
–S33, 2001[Medline]
Smogorzewski MJ: Central nervous dysfunction in uremia.
Am J Kidney Dis 38
[Suppl 1]: S122
–128, 2001[Medline]
Swarthout JT, D’Alonzo RC, Selvamurugan N, Partridge NC: Parathyroid hormone-dependent signaling pathways regulating genes in bone cells.
Gene 282
: 1
–17, 2002[Medline]
Picotto G, Massheimer V, Boland R: Parathyroid hormone stimulates calcium influx and the cAMP messenger system in rat enterocytes.
Am J Physiol 273
: C1349
–C1353, 1997[Medline]
Jansson I, Curti M, Epstein PM, Peterson JA, Schenkman JB: Relationship between phosphorylation and cytochrome P450 destruction.
Arch Biochem Biophys 283
: 285
–292, 1990[CrossRef][Medline]
Pyerin W, Horn F, Taniguchi H: Posttranslational modifications of the cytochrome P-450 monooxygenase system.
J Cancer Res Clin Oncol 113
: 155
–159, 1987[CrossRef][Medline]
Sidhu JS, Omiecinski CJ: cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures.
J Biol Chem 270
: 12762
–12773, 1995[Abstract/Free Full Text]
Galisteo M, Marc N, Fautrel A, Guillouzo A, Corcos L, Lagadic-Gossmann D: Involvement of cyclic nucleotide- and calcium-regulated pathways in phenobarbital-induced cytochrome P-450 3A expression in mouse primary hepatocytes.
J Pharmacol Exp Ther 290
: 1270
–1277, 1999[Abstract/Free Full Text]
LeHoux JG, Dupuis G, Lefebvre A: Regulation of CYP11B2 gene expression by protein kinase C.
Endocr Res 26
: 1027
–1031, 2000[Medline]
Clyne CD, White PC, Rainey WE: Calcium regulates human CYP11B2 transcription.
Endocr Res 22
: 485
–492, 1996[Medline]
Graier WF, Simecek S, Sturek M: Cytochrome P450 mono-oxygenase-regulated signalling of Ca2+ entry in human and bovine endothelial cells.
J Physiol (Lond) 482
: 259
–274, 1995[Medline]
Li Q, Verma IM: NF-kappaB regulation in the immune system.
Nat Rev Immunol 2
: 725
–734, 2002[CrossRef][Medline]
Tian Y, Ke S, Denison MS, Rabson AB, Gallo MA: Ah receptor and NF-kappaB interactions, a potential mechanism for dioxin toxicity.
J Biol Chem 274
: 510
–515, 1999[Abstract/Free Full Text]
Ke S, Rabson AB, Germino JF, Gallo MA, Tian Y: Mechanism of suppression of cytochrome P-450 1A1 expression by tumor necrosis factor-alpha and lipopolysaccharide.
J Biol Chem 276
: 39638
–39644, 2001[Abstract/Free Full Text]
Ali NN, Gilston V, Winyard PG: Activation of NF-kappaB in human osteoblasts by stimulators of bone resorption.
FEBS Lett 460
: 315
–320, 1999[CrossRef][Medline]
Guillen C, Martinez P, de Gortazar AR, Martinez ME, Esbrit P: Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by nuclear factor-kappa B activation in osteoblastic cells.
J Biol Chem 277
: 28109
–28117, 2002[Abstract/Free Full Text]
Bistrian BR: Role of the systemic inflammatory response syndrome in the development of protein-calorie malnutrition in ESRD.
Am J Kidney Dis 32
[Suppl 4]: S113
–S117, 1998[Medline]
Stenvinkel P, Heimburger O, Paultre F, Diczfalusy U, Wang T, Berglund L, Jogestrand T: Strong association between malnutrition, inflammation, and atherosclerosis in chronic renal failure.
Kidney Int 55
: 1899
–1911, 1999[CrossRef][Medline]
Higuchi T, Yamamoto C, Kuno T, Mizuno M, Takahashi S, Kanmatsuse K: Increased production of interleukin-1beta and interleukin-1 receptor antagonist by peripheral blood mononuclear cells in undialyzed chronic renal failure.
Nephron 76
: 26
–31, 1997[Medline]
Akahoshi T, Kobayashi N, Hosaka S, Sekiyama N, Wada C, Kondo H: In-vivo induction of monocyte chemotactic and activating factor in patients with chronic renal failure.
Nephrol Dial Transplant 10
: 2244
–2249, 1995[Abstract/Free Full Text]
Oettinger CW, Bland LA, Oliver JC, Arduino MJ, McAllister SK, Favero MS: The effect of uremia on tumor necrosis factor-alpha release after an in vitro whole-blood endotoxin challenge.
J Am Soc Nephrol 4
: 1890
–1895, 1994[Abstract]
Sunder-Plassmann G, Kapiotis S, Gasche C, Klaar U: Functional characterization of cytokine autoantibodies in chronic renal failure patients.
Kidney Int 45
: 1484
–1488, 1994[Medline]
Ziesche R, Roth M, Papakonstantinou E, Nauck M, Horl WH, Kashgarian M, Block LH: A granulocyte inhibitory protein overexpressed in chronic renal disease regulates expression of interleukin 6 and interleukin 8.
Proc Natl Acad Sci U S A 91
: 301
–305, 1994[Abstract/Free Full Text]
This article has been cited by other articles:
E. Simard, J. Naud, J. Michaud, F. A. Leblond, A. Bonnardeaux, C. Guillemette, E. Sim, and V. Pichette Downregulation of Hepatic Acetylation of Drugs in Chronic Renal Failure
J. Am. Soc. Nephrol.,
July 1, 2008;
19(7):
1352 - 1359.
[Full Text][PDF]
Top
Abstract
Introduction
B Pathway on PTH-Induced Downregulation of Liver CYP3A2
-Actin was detected using a mouse anti-chicken 
) and mRNA expression (
) by hepatocytes incubated for 24 h with various concentrations of 1-34 rat PTH in 10% calf serum. CYP3A2 protein expression was measured by Western blot; specific mRNA were quantified by qPCR. Results of samples incubated with calf serum without added PTH were defined as 100%. Representative blots were shown in insert. *P<0.01 when protein expression was compared with hepatocytes incubated without PTH; 
) in normal rat hepatocytes incubated for 24 h with control or CRF rat sera. Sera were used either nontreated or after preadsorption during 16 h at 4°C with latex beads coated with antibodies specific for rat parathyroid hormone (PTH) to remove PTH. A portion of the depleted sera was supplemented with 10–9 M 1-34 rat PTH before incubation with hepatocytes. CYP3A2 protein expression was measured by Western blot; specific mRNA were quantified by quantiative PCR (qPCR). Results of samples incubated with control rat serum were defined as 100%. Representative blots were shown in insert. *P <0.05 when compared with control (CTL) serum; 
P <0.05 when compared with CTL-PTX rats.
